Plasma variation of corticosteroid-binding globulin and sex hormone-binding globulin.

Sex hormone-binding globulin (SHBG) and corticosteroid-binding globulin (CBG) circulate in plasma and bind their cognate ligands with high affinity, offering a steroid delivery system to target tissues by a variety of mechanisms. Analysis of these steroid-binding proteins is gaining importance in the clinical setting, although more information is warranted on their diurnal and biological variation. This study shows that plasma SHBG (in normal subjects) exhibits little diurnal or biological variation over the 30 day period studied, in contrast to CBG, where plasma levels peak in the early afternoon. This leads to attenuation of the diurnal free cortisol level rhythm compared to total cortisol. We also show that plasma CBG is significantly lower in male subjects with the metabolic syndrome compared to age-matched lean counterparts, and may therefore act as a surrogate marker of insulin resistance. The consequence of lower levels of CBG in these obese male subjects is reflected by higher levels of circulating free cortisol, potentially offering a more favourable environment for adipogenesis.     

Plasma cortisol and corticosteroid-binding globulin in essential hypertension

Plasma concentration of cortisol, total CBG-binding capacity, and blood pressure were measured in control subjects (n = 171), patients with essential hypertension (EH; n = 210) and their first-degree normotensive (NR; n = 84) or hypertensive (HR; n = 66) relatives. Mean (+/- SD) plasma cortisol was significantly (p less than 0.001) decreased in EH (10.1 +/- 4.3 g/dl) patients and HR (11.7 +/- 4.1). Plasma cortisol in NR did not differ from control values (14.3 +/- 4.5) but the distribution of individual values covered the entire control-EH (14.6 +/- 5.5) range. Mean (+/- SD) CBG-binding capacity was significantly (p less than 0.001) lower in EH (14.4 +/- 3.0), NR (17.5 +/- 2), HR (17.6 +/- 2.2) as compared to controls (20.9 +/- 2.1), indicating that the decline in EH and in most relatives was mainly in plasma CBG-bound cortisol. The plasma CBG-binding capacity for cortisol was significantly negatively correlated with mean arterial pressure (MAP) in both controls (p less than 0.001) and NR (p less than 0.01) but not in either HR (r = 0.02) or never-treated EH patients. Total afternoon plasma aldosterone was higher (p less than 0.01 vs. controls) in 93 untreated EH patients (11.2 +/- 4.8 ng/dl) than in either 161 first-degree relatives (8.1 +/- 3.4 ng/dl) or 117 controls (7.6 +/- 3.5 ng/dl). The respective aldosterone-binding globulin (ABG) binding capacities for aldosterone were 21.2 +/- 6.7, 20.1 +/- 9.3 and 9.8 +/- 4.0%. In all these subjects taken together, there was a positive correlation between MAP and ABG-binding capacity (r = 51; p less than 0.001). The association of reduced plasma cortisol and decreased CBG binding capacity in EH may be closely related to altered steroid metabolism, which may be partly explained by an abnormality resembling a relative deficiency in adrenal 17 alpha- and 11 beta-hydroxylation. In some EH patients, hypertension may be the result of the ineffectiveness of plasma cortisol in preventing slightly elevated endogenous ACTH levels leading to an increase in ACTH-sensitive steroids.     

Rabbit corticosteroid-binding globulin: primary structure and biosynthesis during pregnancy

The cDNA-deduced primary structure of rabbit corticosteroid-binding globulin (CBG) contains 383 amino acids (mol wt, 42,326), including three cysteine residues and four sites for N-glycosylation. It is primarily the product of a 1.68-kilobase hepatic mRNA, but small amounts of CBG mRNA were also found in maternal lung, spleen, and ovary and fetal kidney. In the fetus, hepatic CBG mRNA concentrations increase markedly after day 11 and were 2- to 5-fold higher than those in maternal liver during days 17-23. They then declined to very low levels at term (31 days). By contrast, maternal hepatic CBG mRNA levels did not increase until day 23; reached a peak at about day 27, and then declined to prepregnancy values by 3 days after delivery. In general, fetal and maternal hepatic CBG mRNA concentrations reflect the corresponding serum CBG levels. Our data, therefore, indicate that the marked changes in fetal and maternal plasma CBG levels during pregnancy reflect changes in the biosynthesis of the protein rather than alterations in compartmentalization or clearance.     

Structure and function of corticosteroid-binding globulin: Role of carbohydrates

To study the site-specificity of human corticosteroid-binding globulin (CBG) glycosylation and the functional significance of individual carbohydrate chains in its molecule, a panel of recombinant CBG mutants containing each of the six potential glycosylation sites alone and in various combinations has been expressed in Chinese hamster ovary (CHO) cells. Analyses of these mutant glycoproteins showed that three of the glycosylation sites are only partially utilized, and this may contribute to the production of glycoforms with distinct physiological functions. Processing of individual carbohydrate chains (branching and fucosylation) is site-specific and may, thus, account for the formation of structural determinants essential for the recognition of CBG by cell membranes. Glycosylation at the only phylogenetically conserved consensus site, Asn²³⁸-Gly²³⁹-Thr²⁴⁰, is essential for the biosynthesis of CBG with steroid-binding activity. Evidence has been obtained to support the hypothesis that transient carbohydrate-polypeptide interactions between Trp²⁶⁶ and the maturing carbohydrate chain at Asn²³⁸ occur during early stages of the CBG biosynthesis which affect protein folding and formation of the steroid-binding site. Another tryptophan residue, Trp³⁷¹, has been found to be critical for CBG-steroid interactions and is likely located in the steroid-binding site.     

Origin of corticosteroid-binding globulin in fetal rat. Comparative dynamics of corticosteroid-binding globulin, alpha-fetoprotein, and albumin secretion in primary cultures of fetal rat hepatocytes

The origin of corticosteroid-binding globulin (CBG) and its evolution in comparison with alpha-fetoprotein (AFP) and albumin synthesis, during early development of rat liver (days 13 and 15 of fetal life), have been investigated using cultured fetal hepatocytes. Synthesis and secretion of CBG, AFP, and albumin is evidence by cycloheximide-sensitive [14C]leucine incorporation into immunoprecipitable polypeptides secreted by cultured hepatocytes into the medium, two-dimensional immunoelectrophoretic and autoradiographic identification of newly synthesized labeled proteins, corticosterone and estradiol-17 beta binding to CBG and AFP, respectively, and indirect immunofluorescence localization of AFP, albumin, and CBG in cultured fetal hepatocytes. CBG, albumin, and AFP accounted for 6, 11, and 25% (in 13-day-old rat fetuses) and 5, 15, and 28% (15-day-old rat fetuses), respectively, of the total secreted proteins in the culture medium. The rates of CBG, AFP, and albumin (counts/minute of secretion [14C]leucine incorporated per milligram of cell protein/hour of culture) in the hepatocytes of 15-day-old rat fetuses were 1.48-, 2.1-, and 2.57-fold higher, respectively, than in the 13-day-old rat fetuses. These results indicate that fetal liver is also active in CBG synthesis, along with AFP and albumin, as early as day 13 of fetal life and that the synthetic rates of these secretory proteins depend upon the developmental stage of the fetal liver. This developmental related change in the rate of synthesis of CBG by the fetal hepatocytes may regulate the level of free (active) glucocorticoid in the fetal circulation and thereby the initiation and regulation of glucocorticoid-dependent processes during the crucial stages of the differentiation of fetal liver and other developing tissues.     

The S-to-R transition of corticosteroid-binding globulin and the mechanism of hormone release

Corticosteroids are transported in the blood by a serpin, corticosteroid-binding globulin (CBG), and their normally equilibrated release can be further triggered by the cleavage of the reactive loop of CBG. We report here the crystal structures of cleaved human CBG (cCBG) at 1.8-Å resolution and its complex with cortisol at 2.3-Å resolution. As expected, on cleavage, CBG undergoes the irreversible S-to-R serpin transition, with the cleaved reactive loops being fully incorporated into the central β-sheet. A connecting loop of helix D, which is in a helix-like conformation in native CBG, unwinds and grossly perturbs the hormone binding site following β-sheet expansion in the cCBG structure but shifts away from the binding site by more than 8 Å following the binding of cortisol. Unexpectedly, on cortisol binding, the hormone binding site of cCBG adopts a configuration almost identical with that of the native conformer. We conclude that CBG has adapted an allosteric mechanism of the serpins to allow equilibrated release of the hormones by a flip-flop movement of the intact reactive loop into and out of the β-sheet. The change in the hormone binding affinity results from a change in the flexibility or plasticity of the connecting loop, which modulates the configuration of the binding site.     

Purification and partial characterization of a corticosteroid-binding globulin from hamster serum

Our objective was to characterize and purify the corticosteroid-binding proteins in hamster pregnancy serum. When [3H]cortisol-labeled pregnancy and proestrous serum were subjected to native polyacrylamide gel electrophoresis, a single peak of specific steroid-binding activity was detected in each, with identical electrophoretic mobility. The steroid-binding affinity (Ka = 1.07.10(8) M-1 for cortisol) is typical of corticosteroid-binding globulin from other species, but the steroid-binding specificity (cortisol greater than testosterone greater than progesterone) is not. An ultraviolet photoaffinity-labeling protocol was developed using 17 beta-hydroxy-4,6-[1,2-3H]androstadiene-3-one ([3H]androstadienolone), permitting analysis of ultraviolet photoaffinity-labeled proestrous and pregnancy serum by two-dimensional polyacrylamide gel electrophoresis and fluorography. Both sera contained the same labeled protein species. Corticosteroid-binding globulin was purified from pregnancy serum by DEAE-cellulose chromatography followed by steroid affinity chromatography on androstadienolone-17 beta-hemisuccinate-ethylenediamine-AffiGel 10. The purified protein (Mr = 62,250; pI = 3.95; n = 1; Stokes radius = 3.5; S = 4-5) was determined to be a glycoprotein. When analyzed by gel filtration and two-dimensional polyacrylamide gel electrophoresis, purified corticosteroid-binding globulin behaved the same as in unfractionated serum, and when ultraviolet photoaffinity-labeled with [3H]androstadienolone, purified corticosteroid-binding globulin produced the same fluorogram spot pattern seen in unfractionated serum. A specific corticosteroid-binding globulin antiserum was raised in rabbits, and this antiserum reacted with a single spot in Western blots of unfractionated serum. Thus, hamster pregnancy serum was determined to have one corticosteroid-binding protein. This protein is identical to the corticosteroid-binding globulin found in proestrous serum, with a higher titer in pregnancy serum. No other steroid-binding component is observed in hamster serum.     

Hormonal effects on the secretion and glycoform profile of corticosteroid-binding globulin

Corticosteroid-binding globulin (CBG) is a plasma glycoprotein that is primarily synthesized in the liver and binds cortisol and progesterone with high affinity. In this study, a CBG secreting hepatocellular carcinoma derived cell line (HepG2) was used to investigate the hormonal regulation of hepatic CBG synthesis. HepG2 cells were grown for 72 h in 30, 300 and 3000 nM concentrations of estradiol (E₂), testosterone (T), insulin, thyroxin (T₄) and dexamethasone (DMZ) and the secreted CBG quantified by a novel enzyme-linked immunosorbent assay (ELISA). Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) was carried out to determine the effects of these hormones on the relative distribution of CBG glycoforms.

Insulin, T₄ and high concentrations of E₂ decreased the secretion of CBG by HepG2 cells (p < 0.05). Ethanol, the solvent used for E₂, T and DMZ, also significantly attenuated CBG secretion. 2D-PAGE resolved 13–14 glycoforms of CBG produced by HepG2 cells. Insulin caused a reduction in the synthesis of more acidic, while T₄ and DMZ decreased the production of more basic CBG glycoforms. Stimulation with E₂ resulted in the synthesis of additional isoforms of increased acidity, which may represent a type of CBG only seen during pregnancy in vivo. Possible physiological implications of these findings are discussed.     

Pregnancy-associated corticosteroid-binding globulin: high resolution separation of glycan isoforms.

Corticosteroid-binding globulin (CBG) is a glycoprotein that functions as a specific carrier of cortisol in the circulation. CBG contains six sites for N-glycosylation with, on average, five sites occupied by a mixture of biantennary and triantennary oligosaccharides with variable additional terminal sialic acid residues leading to glycoforms with significant heterogeneity in mass and isoelectric points. During pregnancy, a form of CBG possessing only triantennary oligosaccharides comprising approximately 10 % of total CBG appears specifically. We describe the first application of two-dimensional gel electrophoresis to the separation of human CBG glycoforms. This technique resolved a greater degree of charge heterogeneity than previous studies, and allowed simultaneous visualization of changes to the size and isoelectric points of CBG during pregnancy. Profiles of CBG glycoforms during pregnancy showed a general increase in size followed by a shift to lower pI in a large proportion of the glycoprotein. This may result from the enhancement of triantennary glycosylation, with the extent of incorporation of sialic acid increasing with the number of available sites for its addition. The pregnancy-specific CBG previously defined probably represents a subset of the acidic and high molecular weight glycoforms we have resolved by two-dimensional electrophoresis and now describe as pregnancy-associated CBG.     

Kinetic and equilibrium studies on steroid interaction with human corticosteroid-binding globulin

Kinetic and equilibrium studies on the interaction of steroids with human corticosteroid-binding globulin (CBG, transcortin) were performed with pH, temperature, and steroid structure as variables. Dissociation rate constants were determined fluorometrically; the values for cortisol, corticosterone, deoxycorticosterone, and progesterone are 0.031, 0.047, 0.10, and 0.16 s-1, respectively, at 20 degrees C, pH 7.4. The pH dependence of the dissociation rate constant for the corticosterone complex below pH 10.5 at 20 degrees C is given by koff = 0.043 (1 + [H+]/10(-6.50)) s-1; above pH 11, koff = 0.030 (1 + 10(-12.15/[H+] s-1. A temperature-dependence study of koff for the cortisol and progesterone complexes gave values of 0.0028 s-1 and 0.012 s-1 at 4 degrees C, respectively, and 0.88 s-1 and 4.5 s-1 at 37 degrees C, with progesterone dissociating about four to five times faster over the entire temperature range. The affinity constants, determined by equilibrium dialysis, for the binding of cortisol, corticosterone, and progesterone at 4 degrees C were 7.9, 7.2, and 7.0 X 10(8) M-1; values of 0.40 and 0.26 X 10(8) M-1 were determined at 37 degrees C for cortisol and progesterone. The close similarity of the affinity constants of the three steroids combined with differing dissociation rates implies that the association rate changes with steroid structure, in contrast to our earlier findings with progesterone-binding globulin.     

Biosynthesis of hepatic corticosteroid-binding globulin: ontogeny and effect of thyroid hormone.

The present study reports on the ontogeny and the effect of thyroid hormones on liver corticosteroid-binding globulin (CBG) biosynthesis, in relation to plasma CBG binding capacity in the rat. We show that mRNACBG contents were high in liver of 18-day-old fetuses and decreased with age to reach almost undetectable levels by postnatal day 1. Interestingly, at the latter time point plasma CBG concentration remained elevated and disappeared thereafter from the circulation with a half-life of about 3 days. The messenger was localized in parenchymatous cells and not in hematopoietic ones, although the latter constitute the major cell population in fetal liver. It is not until after 10 days of age that mRNACBG and plasma CBG levels increased in concert, with a sex-difference being noticed by postnatal day 30. Treatment of rats with 3,3',5-triiodo-L-thyronine (T3), but not reverse T3 (rT3) (the predominant form present in fetal serum), enhanced CBG biosynthesis. These findings show that liver mRNACBG contents undergo dramatic changes during ontogeny and suggest the existence of a differential regulation of the messenger in fetal and neonatal rats.     

Flow cytometric analysis of DNA replication during the differentiation of 3T3-L1 preadipocytes

We have employed a flow cytometric method of monitoring DNA synthesis in order to study the stimulation of DNA replication during the induction of adipocyte differentiation in the preadipocyte cell line 3T3-L1. When confluent monolayers of this cell line are treated with a mixture of methyl isobutyl xanthine, dexamethasone, and insulin (MDI), they undergo at least one round of cell division, which is followed by the de novo synthesis of many enzymes that are involved in the formation of lipid. If the MDI-treated cells are incubated in iododeoxyuridine (IdUrd)- or bromodeoxyuridine (BrdUrd)-containing medium and subsequently stained with antibodies specific for these base analogues and with propidium iodide (PI), the kinetics of the induction of replication can be monitored. No differences in the patterns of IdUrd incorporation versus PI staining were observed between exponentially growing 3T3-L1 cells and those that had been stimulated to replicate DNA with MDI. In addition, we developed a flow cytometric (FCM) method for staining fatty acid synthetase and localizing the antigen in the G1 phase. We have thus demonstrated the feasibility of this methodology for correlating by FCM the production of enzymes such as fatty acid synthetase with IdUrd and BrUrd incorporation. The technique should permit studies of the inhibition of differentiation of adipocytes by halogenated pyrimidines.     

Rat corticosteroid-binding globulin (CBG) biosynthesis by fetal hepatocytes in culture

The ability of fetal liver to synthesize and secrete corticosteroid-binding globulin (CBG) was investigated using primary cultures of hepatocytes transplanted from 15- and 18-day-old rat fetuses. Analysis of culture medium, after labelling with [14C]leucine, by crossed immunoelectrophoresis, clearly demonstrated CBG, albumin and alpha-fetoprotein (AFP) secretion. The relative rate of CBG secretion was more important at the younger stage. Indirect immunofluorescence permitted localization of CBG in fetal hepatocytes. The results suggest that the high level of CBG found in fetal serum is mainly produced by the liver of the fetus itself.     

Corticosteroid-dependent differentiation of human marrow preadipocytes in vitro

A unique population of human bone marrow-derived, adherent fibroblastlike cells differentiates to adipocyte morphology when grown in vitro in the presence of horse serum and hydrocortisone sodium hemisuccinate. Over the initial 8-weeks growth at 37 degrees C, 7% CO2, these cells accumulate Oil Red O-positive lipid and form colonies of over 100 cells, which persist in confluent cultures for over 30 weeks. Similar to cultures derived from mouse marrow, corticosteroid-induced adipocyte differentiation is associated with long-term granulopoiesis. Human marrow preadipocytes, as well as human, mouse and rat embryo fibroblast cell lines, failed to differentiate to adipocyte morphology in the presence of insulin. In contrast, the 3T3-L1 insulin-dependent preadipocyte cell line was not induced to differentiate in the presence of hydrocortisone. These studies demonstrate that human marrow preadipocytes are dependent upon corticosteroid for differentiation in vitro.     

Purification and characterization of the corticosteroid-binding globulin of pregnant guinea pig serum.

The corticosteroid-binding globulin from guinea pig pregnancy serum was purified by the sequential use of affinity chromatography, hydroxylapatite chromatography, and gel filtration chromatography at a cumulative yield of 80%. The protein was found to be homogeneous by analytical gel electrophoresis, equilibrium sedimentation ultracentrifugation, immunoelectrophoresis, and stoichiometry (1:1) of steroid binding. Guinea pig corticosteroid-binding globulin has a molecular weight of 43 300 and contains 29% carbohydrate. The intrinsic fluorescence of the corticosteroid-binding globulin is quenched by about 73% when 1 mol of cortisol is bound. The association constants (pH 7.4) at 4 and 37 degrees C are 2.5 X 10(7) and 1.5 X 10(6) M-1 for cortisol and 1.4 X 10(6) and 0.2 X 10(6) M-1 for progesterone, respectively.     

Corticosteroid-dependent differentiation of human marrow preadipocytes in vitro

Summary A unique population of human bone marrow-derived, adherent fibroblastlike cells differentiates to adipocyte morphology when grown in vitro in the presence of horse serum and hydrocortisone sodium hemisuccinate. Over the initial 8-weeks growth at 37°C, 7% CO₂, these cells accumulate Oil Red O-positive lipid and form colonies of over 100 cells, which persist in confluent cultures for over 30 weeks. Similar to cultures derived from mouse marrow, corticosteroid-induced adipocyte differentiation is associated with long-term granulopoiesis. Human marrow preadipocytes, as well as human, mouse and rat embryo fibroblast cell lines, failed to differentiate to adipocyte morphology in the presence of insulin. In contrast, the 3T3-L1 insulin-dependent preadipocyte cell line was not induced to differentiate in the presence of hydrocortisone. These studies demonstrate that human marrow preadipocytes are dependent upon corticosteroid for differentiation in vitro. Supported by National Cancer Institute Virus Cancer Program Contract NCI NO1-7-1051.